Mediator subunit MDT-15 promotes expression of propionic acid breakdown genes to prevent embryonic lethality in Caenorhabditis elegans

Abstract The micronutrient vitamin B12 is an essential cofactor for two enzymes: methionine synthase, which plays a key role in the one-carbon cycle; and methylmalonyl-CoA mutase, an enzyme in a pathway that breaks down branched-chain amino acids and odd-chain fatty acids. A second, vitamin B12-independent pathway that degrades propionic acid was recently described in Caenorhabditis elegans, the propionate shunt pathway. Activation of five shunt pathway genes in response to low vitamin B12 availability or high propionic acid levels is accomplished by a transcriptional regulatory mechanism involving two nuclear hormone receptors, NHR-10 and NHR-68. Here, we report that the C. elegans Mediator subunit mdt-15 is also essential for the activation of the propionate shunt pathway genes, likely by acting as a transcriptional coregulator for NHR-10. C. elegans mdt-15 mutants fed with a low vitamin B12 diet have transcriptomes resembling those of wild-type worms fed with a high vitamin B12 diet, with low expression of the shunt genes. Phenotypically, the embryonic lethality of mdt-15 mutants is specifically rescued by diets high in vitamin B12, but not by dietary polyunsaturated fatty acids, which rescue many other phenotypes of the mdt-15 mutants. Finally, NHR-10 binds to MDT-15 in yeast two-hybrid assays, and the transcriptomes of nhr-10 mutants share overlap with those of mdt-15 mutants. Our data show that MDT-15 is a key coregulator for an NHR regulating propionic acid detoxification, adding to roles played by NHR:MDT-15 partnerships in metabolic regulation and pinpointing vitamin B12 availability as a requirement for mdt-15 dependent embryonic development.


Introduction
Animals adjust their metabolism based on their nutritional environment, which allows them to optimize the use of available resources and adjust to or compensate for absent or limiting nutrients. Such metabolic adjustments occur through various mechanisms, including alteration in the expression of metabolic and other genes via transcriptional modulation. Regulation can involve direct sensing of nutritional components or metabolites by transcription factors such as nuclear hormone receptors (NHRs) and indirect sensing via upstream regulators such as G protein-coupled receptors or other sensors (Boukouris et al. 2016;Husted et al. 2017;Schneider-Poetsch and Yoshida 2018;Suganuma and Workman 2018).
Vitamins are a group of essential micronutrients required in many developmental and physiological functions. All animals require vitamin B12, which is vital for DNA synthesis, and fatty acid and amino acid metabolism (Deodato et al. 2017;Froese et al. 2019). Vitamin B12 is an essential cofactor for methylmalonyl-CoA mutase (MUT), an enzyme in the canonical and evolutionarily conserved propionic acid breakdown pathway (Bito and Watanabe 2016;Deodato et al. 2017;Froese et al. 2019). The loss of activity in this pathway due to mutations in the MUT gene or in the genes for propionyl-CoA-carboxylase (PCCA/B) causes the inborn errors of metabolism methylmalonic acidemia and propionic acidemia, respectively (Deodato et al. 2017). Abnormal metabolism in these genetic diseases severely affects development and neurological functions and can cause lethality in newborns.
Recent work in Caenorhabditis elegans has revealed the existence of a parallel, vitamin B12-independent propionic acid breakdown pathway, termed the propionic acid breakdown shunt ('the shunt') (Watson et al. 2016). The shunt is composed of enzymes that are conserved in humans (acdh-1/ACABCB, ech-6/ECHS1, hach-1/HIBCH, hpdh-1/ADHFE1, and alh-8/ALDH6A1) (Watson et al. 2016). However, as intermediate metabolites of the shunt are toxic, shunt gene expression is repressed except when the pathway is absolutely required. In C. elegans, conditions of low dietary vitamin B12 or high dietary propionic acid result in increased shunt gene expression Watson et al. 2013Watson et al. , 2014Watson et al. , 2016.
The tight regulation of shunt genes in C. elegans is achieved by combinatorial activity of NHR-10 and NHR-68 (Bulcha et al. 2019). Both NHRs are required to induce shunt gene expression and for organismal survival on diets with high propionic acid levels. Interestingly, NHR-10 itself activates nhr-68, yet, nhr-68 expression alone is insufficient to induce the propionate shunt genes; rather, nhr-10 and nhr-68 are both required for this activation, and hence, form a self-reinforcing regulatory circuit that only results in gene activation when elevated propionic acid levels persist in the media for several hours (Bulcha et al. 2019).
Like all transcription factors, NHRs do not act in isolation but require coregulators to control gene expression. In C. elegans, the Mediator subunit MDT-15 is a coregulator for many NHRs that regulate nutritional and stress adaptive responses (Grants et al. 2015;Hartman et al. 2021). Accordingly, mdt-15 mutation or depletion results in many developmental and physiological phenotypes, many of which can be rescued by supplementation with unsaturated fatty acids (Taubert et al. 2006;Yang et al. 2006;Hou et al. 2014;Lee et al. 2015Lee et al. , 2019. However, some phenotypes of the mdt-15 mutant cannot be rescued by unsaturated fatty acids, suggesting that other dietary processes and metabolic regulatory pathways may be linked to mdt-15 (Goh et al. 2014(Goh et al. , 2018.
Here, we show that transcriptomic changes caused by mdt-15 loss resemble those of worms grown on a low vitamin B12 diet, with shunt genes downregulated in both conditions. Supplementation with vitamin B12-rich diets, but not with unsaturated fatty acids, rescued the embryonic lethality of mdt-15 mutants. NHR-10, which controls shunt gene expression, physically interacts with MDT-15, and loss of either regulator produces similar transcriptomic perturbations. Our data suggest a model wherein C. elegans MDT-15 acts as a coregulator for NHR-10, with the two factors inducing shunt genes on demand to ensure propionic acid detoxification and prevent adverse organismal phenotypes.

C. elegans strains
Worm strains used in this study were N2 wild type (WT) and XA7702 mdt-15(tm2182) (Taubert et al. 2008); the mutant was backcrossed into our lab N2 strain prior to study. For synchronized worm growths, we isolated embryos by standard sodium hypochlorite treatment. Isolated embryos were allowed to hatch overnight on unseeded NGM plates until the population reached a synchronized state of halted development at L1 stage via shortterm fasting (16-24 hours). Synchronized L1 stage larvae were then transferred to seeded plates and grown to the desired stage. WT N2 and XA7702 mdt-15(tm2182) worms were grown to young adults on NGM plates seeded with E. coli OP50 or C. aquatica DA1877. Then, 10 worms were transferred onto plates seeded with E. coli OP50 or C. aquatica DA1877 and left at 20°C for 24 hours. Adults were then removed, and progeny were allowed to hatch for 48 hours. Eggs that had not hatched thereafter were classified as nonviable.

Oxidative stress sensitivity assays
To assess oxidative stress sensitivity, synchronized N2 and mdt-15(tm2182) L1 stage worms were allowed to grow on NGM plates containing 300 μM PUFAs and/or 5 nM vitamin B12 until they reached the mid-L4 stage. Then, they were transferred to plates also containing 2 or 4 mM tBOOH, which were seeded with heat-inactivated E. coli OP50. After 24 hours, the number of dead or alive worms was counted. In parallel, to ascertain PUFA effectiveness, we also scored whether PUFA supplementation was able to rescue viability of N2 on RNAi plates seeded with HT115 bacteria containing control empty vector (EV) of fat-6 RNAi clones.

RNA isolation and RT-qPCR analysis
Synchronized L1 worms were allowed to grow on E. coli OP50 plates for 48 hours to L4 stage and rapidly harvested. RNA isolation was performed as previously described (Doering et al. 2022). Briefly, 2 μg total RNA was used to generate cDNA with Superscript II reverse transcriptase (Invitrogen 18064-014), random primers (Invitrogen 48190-011), dNTPs (Fermentas R0186), and RNAseOUT (Invitrogen 10777-019). Quantitative PCR was performed in 10 μl reactions using Fast SYBR Master Mix (Life Technologies 4385612), 1:10 diluted cDNA, and 5 μM primer, and analyzed with an Applied Biosystems StepOnePlus machine. We analyzed the data with the ΔΔCt method. For each sample, we calculated normalization factors by averaging the (sample expression)/(average reference expression) ratios of three normalization genes, act-1, tba-1, and ubc-2. The reference sample was wild type (WT) grown on E. coli OP50. We used one-way or two-way ANOVA to calculate statistical significance of gene expression changes and corrected for multiple comparisons using the Tukey method. Primers were tested on serial cDNA dilutions and analyzed for PCR efficiency prior to use. All data originate from three or more independent biological repeats, and each PCR reaction was conducted in technical duplicate. Sequences of RT-qPCR primers are listed in Table 1.

Microarray analysis
Microarray gene expression profiling of mdt-15(tm2182) mutants was performed at the University of California San Francisco SABRE Functional Genomics Facility exactly as previously described (Grants et al. 2016), using the same growth conditions, and RNA extraction, processing, hybridization, and analysis methods. Microarray data of mdt-15(tm2182) mutants and corresponding WT are deposited in Gene Expression Omnibus (GEO) under a new accession number (GSE220955), with the WT control exactly the same as previously published [GEO Series GSE68520 (Grants et al. 2016)]. Microarray data were processed using limma (Ritchie et al. 2015) exactly as described (Grants et al. 2016). To maximize comparability of the microarray to RNA-seq datasets, probe identifiers were converted to gene symbols using the ID conversion module of easyGSEA in the eVITTA toolbox (Cheng et al. 2021), and only the highest expressing probe (based on aveA average expression) was kept for each unique gene symbol. Using this approach, we identified 1,616 unique differentially expressed genes (DEGs) with Adj.P < 0.05 in mdt-15(tm2182) mutants, including 563 downregulated (logFC < 0) and 1,053 upregulated (logFC > 0) genes (Supplementary Tables 1 and 2).

Reanalysis of published microarray data
To maximize comparability with our microarray samples, we reanalyzed published two-channel microarray data of mdt-15(RNAi) worms (Taubert et al. 2008). Raw data were extracted from GEO under the accession number GSE9720 and reanalyzed with limma. Adaptive background correction was performed with the method "normexp" with offset 50, and subsequently normalizeWithinArrays using default method (print tip loess). The command modelMatrix with set refere nce level "control RNAi" was used to make the sign of M consistent across repeats regardless of dye swapping. Differentially expressed genes were computed using lmFit, eBayes, and topTable with the de fault parameters, and probe identifiers were converted to unique ge ne symbols as above. We identified 2,451 DEGs with P < 0.05, including 1,298 downregulated and 1,153 upregulated genes (Supplementary  Tables 1 and 3).
We also reanalyzed the published single-channel microarray of Comamonas DA1877 vs E. coli HT115 treatment , for which normalized count data are available in GEO (accession number: GSE43959). We performed data extraction and differntial expression (DE) analysis using easyGEO in the eVITTA toolbox (Cheng et al. 2021), using default parameters (limma with quantile normalization and linear model fitting with ls). Probe identifiers were converted to unique gene symbols as above. We identified 6,541 DEGs with P < 0.05, including 2,979 downregulated and 3,562 upregulated genes (Supplementary Tables 1 and 4).

Reanalysis of published RNA-seq data
To maximize comparability with our microarray samples, we reanalyzed published RNA-sequencing data of Vitamin B12-treated WT worms, and of nhr-10 and nhr-68 mutants (Bulcha et al. 2019). Relevant Sequence Read Archive (SRA) accession numbers and metadata were obtained from SRA run selector under the GEO accession number GSE123507. For each SRA accession number, raw reads were downloaded from SRA using prefetch, and FASTQ files were extracted using fastq-dump. Because the study was done on BGI-seq-500 platform, we compiled a list of adaptor sequences using the overrepresented sequences from FastQC. Following this, the reads were trimmed using Trimmomatic version 0.36 (Bolger et al. 2014) with parameters LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36. Next, trimmed reads were aligned to the NCBI reference genome WBcel235 WS277 (https:// www.ncbi.nlm.nih.gov/assembly/GCF_000002985.6/) using Salmon version 0.9.1 (Patro et al. 2017) with parameters -l A -gcBias -validateMappings. Then, transcript-level read counts were imported into R and summed into gene-level read counts using tximport (Soneson et al. 2016). Genes not expressed at a level greater than one count per million reads in at least two of the samples were excluded from further analysis. The gene-level read counts were normalized using the trimmed mean of M-values in edgeR (Robinson et al. 2010) to adjust samples for differences in library size. Differential expression analysis was performed using the quasi-likelihood F-test with the generalized linear model approach in edgeR (Robinson et al. 2010

Comparison and visualization of transcriptome data
As quality controls, we compared genes deregulated in mdt-15(tm2182) mutants to genes deregulated in mdt-15(RNAi) worms (GSE9720), revealing a substantial correlation between the two datasets ( Supplementary Fig. 1a), as expected. The scatter plot was generated using plotly, and Pearson correlation coefficient was computed using the cor.test function.
To visualize the downregulation of shunt genes in different datasets, we generated volcano plots using limma function plotWithHighlights. We separated DEGs (defined as the genes with Adj.P or P < 0.05) above the line, and shunt genes were marked using additional text labels.
To compare our microarray data to previously published data, we generated Venn diagrams using the R package eulerr (Larsson and Gustafsson 2018). Hypergeometric P-value was calculated using the phyper function, where q = size of overlap 1, m = number of genes in gene list 1, n = platform size m, and k = number of genes in gene list 2. For each pairwise comparison, platform size was calculated as the total number of genes that are detected in DE analysis in either sample.
For overrepresentation analysis (ORA) of gene list overlaps, we analyzed lists of downregulated genes (Adj.P or P < 0.05, and logFC < 0) using the ORA module of easyGSEA in the eVITTA toolbox (Cheng et al. 2021). The following gene set databases were selected for ORA analysis: Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome Pathway (RA), WikiPathways (WP), WormCat Category 2 (C2), WormCat Category 3 (C3), and Gene Ontology: Biological Processes (BP). Top 10 significantly enriched gene sets (P < 0.005 and Adj.P < 0.25) were visualized using the bar plot module. The shunt genes in each gene set are annotated in the plots.
To identify mdt-15 dependent processes that may promote PUFA-independent embryonic development, we compared transcriptome profiles of mdt-15(RNAi) and mdt-15(tm2182) hypomorph mutants to other transcriptomes. We found that the transcriptomes of WT worms fed with the bacterial food source C. aquatica DA1877 (MacNeil et al. 2013) shared a statistically significant overlap with genes dependent on mdt-15 (Fig. 1a). Vitamin B12 (aka cobalamin) is a key molecule that drives C. aquatica-induced developmental acceleration and it is virtually undetectable in E. coli OP50 (Watson et al. 2014). Accordingly, the transcriptomes of worms grown on vitamin B12 supplemented OP50 (Bulcha et al. 2019) also shared a statistically significant overlap with genes dependent on mdt-15 (Fig. 1b). Thus, the loss of mdt-15 results in transcriptomes that partially resemble those of worms fed with vitamin B12-rich diets.

A diet of C. aquatica rescues the embryonic lethality of mdt-15(tm2182) mutants
To determine if a diet of C. aquatica DA1877 might influence the phenotypes of mdt-15(tm2182) mutants, we compared mdt-15(tm2182) mutants fed with C. aquatica DA1877 or E. coli OP50. We observed a substantial increase in the number of viable mdt-15(tm2182) mutant offspring when provided with C. aquatica DA1877 (Fig. 2a). To determine whether maternal or offspring food source caused the phenotypic rescue, we grew mdt-15(tm2182) mutants to adulthood on either E. coli OP50 or C. aquatica DA1877, switched them to the identical or reciprocal food sources, and then quantified the number of surviving offspring. We found that adult mdt-15(tm2182) mutants raised on E. coli OP50 generated mostly arrested progeny even when switched to C. aquatica DA1877, whereas the progeny of worms raised on C. aquatica DA1877 mostly hatched successfully even when the adults were switched to an E. coli OP50 diet (Fig. 2b). Thus, maternal C. aquatica DA1877 is sufficient to rescue the embryonic lethality in mdt-15(tm2182) progeny.

Vitamin B12 rescues the embryonic lethality in mdt-15(tm2182) mutants
Vitamin B12 is the key component of C. aquatica-induced developmental acceleration (Watson et al. 2014). To determine whether vitamin B12 underlies the C. aquatica-induced rescue of the embryonic lethality of mdt-15(tm2182) mutants, we grew them on plates supplemented with 50 nM Me-Cbl, one of the active forms of vitamin B12 (Froese et al. 2019). Me-Cbl supplementation strongly rescued the embryonic lethality of mdt-15(tm2182) mutants (Fig. 2c). Supplementation with 5 or 500 nM of Me-Cbl had a similar effect, and supplementation with 5, 50, or 500 nM Ado-Cbl, another active form of vitamin B12, also effectively rescued embryonic lethality at all concentrations (Fig. 2d). These data show that vitamin B12 levels are essential for normal embryonic viability of mdt-15 mutants.

mdt-15(tm2182) mutants are sensitive to propionic acid
In C. elegans, vitamin B12 is required as a cofactor for two enzymes: the methionine synthase metr-1; and the MUT-type isomerase mmcm-1 (Watson et al. 2016). We reasoned that methionine is unlikely to be in short supply in C. elegans strains feeding on E. coli as food source and thus did not study methionine metabolism. The other C. elegans enzyme that utilizes vitamin B12 as a cofactor is mmcm-1, which is required to clear propionic acid, a toxic intermediate in the catabolism of odd-chain fatty acids and branchedchain amino acids (Watson et al. 2016). To determine whether mdt-15(tm2182) mutants are sensitive to propionic acid, we placed L4 WT and mdt-15 mutant worms grown with or without Me-Cbl on varying concentrations of propionic acid and monitored their survival after 24 hours. Almost 100% of WT worms survived in the presence of 20 mM propionic acid, whether or not supplemented with Me-Cbl; in contrast, <2.5% of mdt-15(tm2182) mutants survived in the presence of 20 mM propionic acid (Fig. 2e). At concentrations between 10 and 20 mM propionic acid, mdt-15(tm2182) mutants showed intermediate viability that was significantly rescued by the addition of Me-Cbl (Fig. 2e). Thus, the loss of mdt-15 renders C. elegans sensitive to propionic acid, and this can be rescued by increasing the activity of vitamin B12 dependent propionic acid degradation pathway.

Unsaturated fatty acids do not rescue the embryonic lethality of mdt-15 mutants
mdt-15 is required to express genes for unsaturated fatty acid synthesis (Taubert et al. 2006;Yang et al. 2006;Hou et al. 2014). To determine whether lack of unsaturated fatty acids contributes to the embryonic lethality phenotype of mdt-15(tm2182) mutants, we grew them on plates containing unsaturated fatty acids, vitamin B12, or a combination of both. Unlike vitamin B12, unsaturated fatty acids did not improve embryonic lethality (Fig. 3a). Fatty acid supplementation was effective in these experiments, as it rescued the reduced brood size of fat-6(RNAi) worms (Fig. 3b), as published (Yang et al. 2006).
Besides embryonic lethality, the oxidative stress sensitivity of mdt-15(tm2182) mutants is also not rescued by dietary supplementation with PUFAs (Goh et al. 2014). We therefore tested whether supplementation with PUFAs, vitamin B12, or a combination of both could rescue the sensitivity of mdt-15(tm2182) mutants to the pro-oxidant tBOOH. As before (Goh et al. 2014), PUFA supplementation had no effects. Similarly, Me-Cbl supplementation, either alone or in combination with PUFAs, failed to increase tBOOH resistance in mdt-15(tm2182) mutant worms (Fig. 3c), suggesting that this phenotype is not related to vitamin B12 dependent metabolism.

mdt-15 is required to express enzymes in the propionic acid shunt breakdown pathway
Propionic acid is degraded via two pathways in C. elegans: the "canonical", vitamin B12 dependent pathway and the shunt pathway that is activated and critical when vitamin B12 is low or unavailable (Watson et al. 2016). Notably, four of five shunt pathway enzymes (acdh-1, ech-6, hach-1, and hphd-1), which are repressed when C. elegans has sufficient vitamin B12 Watson et al. 2014), require mdt-15 for expression on E. coli OP50 (Fig. 1b and Supplementary Fig. 1b and c). We used RT-qPCR to quantify the expression of these genes in WT worms fed with E. coli OP50, WT worms fed with C. aquatica DA1877, and mdt-15(tm2182) mutants fed with E. coli OP50. This confirmed that C. aquatica DA1877 strongly downregulated the expression of acdh-1, hphd-1, hach-1, and ech-6, as expected Watson et al. 2014); in addition, we found that loss of mdt-15 also downregulated acdh-1, hphd-1, hach-1, and ech-6 (Fig. 4a). Thus, mdt-15 is required to activate the expression of some shunt pathway genes in conditions of low vitamin B12 activity.

MDT-15 and NHR-10 coregulate propionic acid shunt breakdown genes
NHR-10 and NHR-68 regulate propionic acid shunt breakdown genes (Bulcha et al. 2019). We previously showed that NHR-68  Diets rich in vitamin B12 rescue the embryonic lethality of mdt-15(tm2182) mutants. a) The graph shows the average embryonic lethality of WT and mdt-15(tm2182) mutant worms grown on either E. coli OP50 or C. aquatica D1877 (n = 3-5). ****P < 0.001 for indicated comparisons, all other comparisons not significant, as assessed by two-way ANOVA with Tukey correction for multiple comparisons. b) The graph shows the average embryonic lethality of mdt-15(tm2182) mutant worms grown on either E. coli OP50 or C. aquatica D1877 for the P0 and F1 generations, as indicated (n = 3). ****P < 0.001 for indicated comparisons, all other comparisons not significant, as assessed by two-way ANOVA with Tukey correction for multiple comparisons. c) The graph shows the average embryonic lethality of WT and mdt-15(tm2182) mutant worms grown on either E. coli OP50, either unsupplemented or supplemented with methyl-cobalamin (Me-Cbl; n = 5). ****P < 0.001 for indicated comparisons, all other comparisons not significant, as assessed by two-way ANOVA with Tukey correction for multiple comparisons. d) The graph shows the average embryonic lethality of mdt-15(tm2182) mutant worms grown on either E. coli OP50, either unsupplemented or supplemented with Me-Cblor adenosyl-cobalamin (Ado-Cbl) at the indicated concentrations (n = 4). ****P < 0.001 for indicated comparisons, all other comparisons not significant, as assessed by ordinary one-way ANOVA with Tukey correction for multiple comparisons. e) The graph shows the average embryonic lethality of WT (left) or mdt-15(tm2182) mutant (right) worms grown on E. coli OP50 on the indicated propionic acid concentrations and either unsupplemented or supplemented with methyl-cobalamin, as indicated (n = 3-4). *P < 0.05 and **** P < 0.001 for the indicated comparisons, as assessed by two-way ANOVA with Tukey correction for multiple comparisons.
does not bind MDT-15 in yeast two-hybrid (Y2H) assays (Taubert et al. 2006). In contrast, we and others detected NHR-10 binding to MDT-15 in Y2H screens (Arda et al. 2010;Reece-Hoyes et al. 2013). Quantification of binding using Y2H assays revealed that an NHR-10 bait protein indeed strongly bounds to several MDT-15 prey proteins (Fig. 4b). Moreover, a construct containing only the KIX-domain of MDT-15 (aa 1-124), previously characterized as an NHR binding domain in MDT-15 (Taubert et al. 2006;Goh et al. 2014;Shomer et al. 2019), was sufficient to mediate this interaction (Fig. 4b). Overlap of gene sets dependent on NHR-10 and NHR-68 (Bulcha et al. 2019) and MDT-15 (this study) revealed that the genes controlled by each of these transcriptional regulators overlap substantially ( Fig. 4c and Supplementary  Table 8), with the shunt genes amongst the most strongly downregulated genes in all of the mutants of these transcriptional regulators ( Supplementary Fig. 1d and e). ORA of gene ontology terms revealed that the genes induced by all three transcriptional regulators as well as genes only coregulated by MDT-15 and NHR-10 are enriched for terms related to lipid and amino acid degradation, reflecting the presence of the shunt genes ( Fig. 4d and e and Supplementary Table 9). ORA of the genes coregulated by MDT-15 and NHR-68 revealed additional enrichment of gene sets related to detoxification (Fig. 4f and Supplementary  Table 9). Overall, our data suggest that MDT-15 and NHR-10 likely interact physically and functionally to control the expression of propionic acid shunt breakdown and other genes in vivo, acting upstream of NHR-68.

Discussion
Vitamin B12 is an essential cofactor that is only synthesized by some species of bacteria. In contrast to the E. coli OP50 diet normally fed to C. elegans in the laboratory, a diet of C. aquatica DA1877 contains higher levels of vitamin B12, which influences a number of life history traits in worms, including brood size, developmental rate, and life span. Here, we show that vitamin B12 is essential for embryonic viability in C. elegans worms carrying a mutation in the mdt-15 gene. Dissecting this requirement further, we find that mdt-15 is required for the activation of genes in the propionic acid degradation shunt, which acts in parallel to the canonical vitamin B12-dependent propionic acid degradation pathway. Binding analysis with Y2H assays and comparison of a) The graph shows the average embryonic lethality of WT and mdt-15(tm2182) mutant worms grown on either E. coli OP50, either unsupplemented or supplemented with Me-Cbl, PUFAs, or Me-Cbl and PUFAs (n = 4-5). ****P < 0.001 for indicated comparisons, all other comparisons not significant, as assessed by two-way ANOVA with Tukey correction for multiple comparisons. b) The graph shows the number of progeny laid by fat-6(RNAi) worms with and without PUFA supplementation (n = 3). ***P < 0.001, unpaired Student's t-test. c) The graph shows the average number of WT and mdt-15(tm2182) mutant worms that are alive after growth on 2 mM or 4 mM tBOOH and supplemented with Me-Cbl, PUFAs, or Me-Cbl and PUFAs, as indicated (n = 3). ****P < 0.001 for indicated comparisons, ns = not significant, two-way ANOVA with Tukey correction for multiple comparisons. mutant transcriptomes suggest that MDT-15 may interact with NHR-10 to regulate shunt gene expression, thus adapting C. elegans metabolism in conditions where propionic acid degradation through the canonical vitamin B12-dependent mechanisms is not possible.
Mutation of nhr-10 or nhr-68 causes larval arrest/delay when propionic acid is present in the media (Bulcha et al. 2019), phenocopying the propionic acid sensitivity of larval development seen after mutation/depletion of mdt-15 or the shunt genes. Curiously, however, nhr-10 or nhr-68 mutation or depletion was not found to cause embryonic or larval arrest (Gönczy et al. 2000;Kamath et al. 2003;Simmer et al. 2003;MacNeil et al. 2013;Bulcha et al. 2019). Bulcha et al. (2019) observed that acdh-1 expression in nhr-10 and nhr-68 mutants is reduced, but not completely abrogated; possibly, mdt-15 mutation lowers the expression of acdh-1 (and other shunt genes) below the levels required for embryonic development. This could be because MDT-15 acts as a coregulator for one (or more) of the 44 other transcription factors that regulate acdh-1 (and perhaps, other shunt genes) (Macneil et al. 2015). These include NHR-114 and SBP-1, which bind MDT-15 (Yang et al. 2006;Arda et al. 2010) and impinge on shunt gene activity (Giese et al. 2020;Qin et al. 2022). Further work will be required to delineate the details of this regulation.
We did not test whether the other vitamin B12 activated pathway, methionine synthesis, is affected by mdt-15 mutation. We reasoned that even if methionine synthesis was low, these worms should receive an adequate methionine supply from their E. coli diet. However, methionine synthesis is linked to the one-carbon cycle, in particular, the metabolism of folate to its biologically active form, tetrahydrofolate. Interestingly, mdt-15 mutants express lower levels of the folate transporter folt-2, as determined by microarray and RT-qPCR analysis (Taubert et al. 2008). However, no overt phenotypes have been reported for folt-2 depletion by RNAi (Gönczy et al. 2000;Simmer et al. 2003;Sönnichsen et al. 2005), perhaps because folt-1 and folt-3 compensate for folt-2 loss.
Our study adds to the growing list of specific gene regulatory programs that are implemented by partnerships between MDT-15 and NHRs (and other TFs). For example, we and others showed that NHR-49 and MDT-15 interact physically and regulate the expression of numerous fatty acid metabolism genes, which in turn affects numerous phenotypes of mdt-15 mutants, including viability, life span, fecundity, mobility, and others (Taubert et al. 2006;Yang et al. 2006). Recent studies suggest that NHR-49 and MDT-15 also interact to promote resistance to several stresses, including starvation, oxidative stress, hypoxia, and pathogen resistance (Goh et al. 2018;Hu et al. 2018;Dasgupta et al. 2020;Hummell et al. 2021;Wani et al. 2021;Doering et al. 2022). MDT-15 also binds NHR-86 (Reece-Hoyes et al. 2013), and both factors regulate the expression of innate immune response genes and ensure survival in response to infection with the pathogen Pseudomonas aeruginosa, with NHR-86 directly sensing the presence of pathogen-derived toxic metabolite (Peterson et al. 2019(Peterson et al. , 2023. Furthermore, an interaction between MDT-15 and HIZR-1 (aka NHR-33) promotes the expression of heavy metal response genes through the high-zinc activated regulatory element Shomer et al. 2019). The data presented here suggest that NHR-10 and MDT-15 interact to transcriptionally induce propionic acid degradation shunt genes when propionic acid levels are high, and/or vitamin B12 levels are low. Interestingly, HIZR-1 directly binds the essential metal zinc as well as nonessential, toxic cadmium (Warnhoff et al. 2017;Earley et al. 2021), and HIZR-1 binding to MDT-15 is strongly increased in the presence of such ligands (Shomer et al. 2019). Possibly, metabolites linked to the propionic acid degradation pathway could similarly be ligands for NHR-10 (Bulcha et al. 2019), although its very short chain length distinguishes it from other such molecules that serve as NHR ligands. It is also possible that upstream metabolites, such as branched-chain amino acids and longer odd-chain fatty acids, act as ligands for NHR-10. Alternatively, the MDT-15-NHR-10 interaction may be altered by posttranslational modifications, or due to NHR-10 dimerizing with a different partner, e.g. forming an NHR-10 dimer that interacts more strongly with MDT-15.
In summary, we report a novel interaction between the gene encoding the transcriptional regulator mdt-15 and vitamin B12, an essential micronutrient. We hypothesize that when vitamin B12 levels are low, and/or propionic acid levels are high, NHR-10 and MDT-15 interaction is stimulated, which in turn allows increased expression of the shunt genes and permits normal embryonic development despite low vitamin B12 availability. It will be interesting to determine whether this mechanistic role for MDT-15 and HNF4-like nuclear receptors is conserved in mammals, especially in view of shunt gene activation by exogenous propionic acid in cultured liver cancer cell lines (Watson et al. 2016).

Data availability
The data underlying this article are available in the article, in its online Supplementary material, and at Gene Expression Omnibus online (GSE220955). All reagents are available upon request.
Supplementary material is available at G3 online.

Conflicts of interest statement
The author(s) declare no conflict of interest.